Genome-wide analysis of basic helix-loop-helix (bHLH) transcription factors in petunia and identification of the putative candidate member involved in floral volatile benzenoids/phenylpropanoids metabolism.

The basic helix-loop-helix (bHLH) family, a prominent group of transcription factors, is involved in plant growth, development, and secondary metabolic processes. Petunia (Petunia hybrida), a beloved and widely cultivated garden flower, boasts a diverse array of varieties, some of which exude a captivating fragrance that has garnered immense popularity. The aromatic allure of petunias primarily stems from the presence of volatile benzenoids/phenylpropanoids, the principal floral scent compounds. But whether bHLH transcription factors regulate petunia floral scent compound synthesis is not clear. In this study, we sought to screen the putative candidate member of bHLH which can be involved in the biosynthesis of benzenoids/phenylpropanoids by examining 63 members of the petunia bHLH gene family. Phylogenetic analysis of the 63 petunia bHLH proteins them into 16 subgroups. Almost all bHLH members contained alkaline/helix-loop-helix domains. Based on the reported RNA sequencing data of P. hybrida 'Mitchell', 30 assembled sequences were mapped to the bHLH genes of P. axillaris. Further qRT-PCR assays suggested that PhbHLH19 might be the putative candidate member in the biosynthesis of benzenoids/phenylpropanoids. PhbHLH19 showed higher expression levels in the petal limb but was lowly expressed at the bud stage, with a rapid increase in the expression level when flowers opened. The expression of PhbHLH19 displayed a significant positive correlation with that of PhPAL2, and the yeast one-hybrid assay verified that PhbHLH19 can bind to the promoter of PhPAL2. Moreover, a dual-luciferase assay proved the transcriptional activation of PhbHLH19 on PhPAL2. These findings suggested that PhbHLH19 might be a putative candidate in the regulation of benzenoid/phenylpropanoid synthesis by activating PhPAL2 expression.